Approximately 10% of humans with anophthalmia (absent eye) or severe microphthalmia (small eye) show haploid insufficiency due to mutations in SOX2, a SOXB1-HMG box transcription factor. However, at present, the molecular or cellular mechanisms responsible for these conditions are poorly understood. Here, we directly assessed the requirement for SOX2 during eye development by generating a gene-dosage allelic series of Sox2 mutations in the mouse. The Sox2 mutant mice display a range of eye phenotypes consistent with human syndromes and the severity of these phenotypes directly relates to the levels of SOX2 expression found in progenitor cells of the neural retina. Retinal progenitor cells with conditionally ablated Sox2 lose competence to both proliferate and terminally differentiate. In contrast, in Sox2 hypomorphic/null mice, a reduction of SOX2 expression to <40% of normal causes variable microphthalmia as a result of aberrant neural progenitor differentiation. Furthermore, we provide genetic and molecular evidence that SOX2 activity, in a concentration-dependent manner, plays a key role in the regulation of the NOTCH1 signaling pathway in retinal progenitor cells. Collectively, these results show that precise regulation of SOX2 dosage is critical for temporal and spatial regulation of retinal progenitor cell differentiation and provide a cellular and molecular model for understanding how hypomorphic levels of SOX2 cause retinal defects in humans.[Keywords: SOX2; allelic series; retinal progenitor identity; dosage regulation; anopthalmia, microapthalmia] Supplemental material is available at www.genesdev.org. It has recently been shown that mutations in SOX2 a SOXB1-HMG box transcription factor whose expression universally marks neural stem and progenitor cells throughout the CNS including the neural retina (Collignon et al. 1996;Zappone et al. 2000;D'Amour and Gage 2003;Ellis et al. 2004;Ferri et al. 2004), are associated with retinal and ocular malformations in humans. The resulting haploid insufficiency at the SOX2 locus occurs in ∼10% of human individuals with anophthalmia or severe microphthalmia (Fantes et al. 2003;Fitzpatrick and van Heyningen 2005;Hagstrom et al. 2005; Ragge et al. 2005a,b;Zenteno et al. 2005). Most mutations identified to date are point mutations leading to truncations of SOX2, while a smaller class of mutations includes microdeletions and missense point mutations. Interestingly, all mutations produce hypomorphic conditions, where residual SOX2 expression and function are still preserved, albeit at lower levels, leading to the highly variable severity of the clinical phenotype. In this regard, the SOX2 mutations in humans and the clinical consequence of reduced functional levels of SOX2 suggest a dosage-dependent role for SOX2 during retinal progenitor differentiation.To date, the importance of SOX2 in the nervous system has been highlighted by misexpression and domi-

Caspase-3 is a major cell death effector protease in the adult and neonatal nervous system. We found a greater number and higher density of cells in the cortex of caspase-3 ؊/؊ adult mice, consistent with a defect in developmental cell death. Caspase-3 ؊/؊ mice were also more resistant to ischemic stress both in vivo and in vitro. After 2 h of ischemia and 48 h of reperfusion, cortical infarct volume was reduced by 55%, and the density of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells was decreased by 36% compared with wild type. When subjected to oxygen-glucose deprivation (2 h), cortical neurons cultured from mice deficient in caspase-3 expression were also more resistant to cell death by 59%. Mutant brains showed caspase-specific poly-(ADP-ribose) polymerase cleavage product (85-kDa fragment) in vivo and in vitro, suggesting redundant mechanisms and persistence of caspase-mediated cell death. In the present study, we found that caspase-8 mediated poly(ADP-ribose) polymerase cleavage in caspase-3 ؊/؊ neurons in vivo and in vitro. In addition, mutant neurons showed no evidence of compensatory activation by caspase-6 or caspase-7 after ischemia. Taken together, these data extend the pharmacological evidence supporting an important role for caspase-3 and caspase-8 as cell death mediators in mammalian cortex and indicate the potential advantages of targeting more than a single caspase family member to treat ischemic cell injury.

Parkinson's disease (PD) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity are both associated with dopaminergic neuron death in the substantia nigra (SN). Apoptosis has been implicated in this cell loss; however, whether or not it is a major component of disease pathology remains controversial. Caspases are a major class of proteases involved in the apoptotic process. To evaluate the role of caspases in PD, we analyzed caspase activation in MPTP-treated mice, in cultured dopaminergic cells, and in postmortem PD brain tissue. MPTP was found to elicit not only the activation of the effector caspase-3 but also the initiators caspase-8 and caspase-9, mitochondrial cytochrome c release, and Bid cleavage in the SN of wild-type mice. These changes were attenuated in transgenic mice neuronally expressing the general caspase inhibitor protein baculoviral p35. These mice also displayed increased resistance to the cytotoxic effects of the drug. MPTP-associated toxicity in culture was found temporally to involve cytochrome c release, activation of caspase-9, caspase-3, and caspase-8, and Bid cleavage. Caspase-9 inhibition prevented the activation of both caspase-3 and caspase-8 and also inhibited Bid cleavage, but not cytochrome c release. Activated caspase-8 and caspase-9 were immunologically detectable within MPP(+)-treated mesencephalic dopaminergic neurons, dopaminergic nigral neurons from MPTP-treated mice, and autopsied Parkinsonian tissue from late-onset sporadic cases of the disease. These data demonstrate that MPTP-mediated activation of caspase-9 via cytochrome c release results in the activation of caspase-8 and Bid cleavage, which we speculate may be involved in the amplification of caspase-mediated dopaminergic cell death. These data suggest that caspase inhibitors constitute a plausible therapeutic for PD.

Recent studies have demonstrated that the topography of thalamocortical (TC) axon projections is initiated before they reach the cortex, in the ventral telencephalon (VTel). However, at this point, the molecular mechanisms patterning the topography of TC projections in the VTel remains poorly understood. Here, we show that a long-range, high-rostral to low-caudal gradient of Netrin-1 in the VTel is required in vivo for the topographic sorting of TC axons to distinct cortical domains. We demonstrate that Netrin-1 is a chemoattractant for rostral thalamic axons but functions as a chemorepulsive cue for caudal thalamic axons. In accordance with this model, DCC is expressed in a high-rostromedial to low-caudolateral gradient in the dorsal thalamus (DTh), whereas three Unc5 receptors (Unc5A–C) show graded expression in the reverse orientation. Finally, we show that DCC is required for the attraction of rostromedial thalamic axons to the Netrin-1–rich, anterior part of the VTel, whereas DCC and Unc5A/C receptors are required for the repulsion of caudolateral TC axons from the same Netrin-1–rich region of the VTel. Our results demonstrate that a long-range gradient of Netrin-1 acts as a counteracting force from ephrin-A5 to control the topography of TC projections before they enter the cortex.

Cell death from spinal cord injury is mediated in part by apoptotic mechanisms involving downstream caspases (e.g., caspase-3). Upstream mechanisms may involve other caspases such as procaspase-8, a 55 kDa apical caspase, which we found constitutively expressed within spinal cord neurons along with Fas. As early as 1.5 hr after transient ischemia, activated caspase-8 (p18) and caspase-8 mRNA appeared within neurons in intermediate gray matter and in medial ventral horn. We also detected evidence for an increase in death receptor complex by co-immunoprecipitation using Fas and anti-procaspase-8 after ischemia. At early time points, Fas and p18 were co-expressed within individual neurons, as were activated caspase-8 and caspase-3. Moreover, we detected p18 in cells before procaspase-3 cleavage product (p20), suggesting sequential activation. The appearance of cytosolic cytochrome c and gelsolin cleavage after ischemia was consistent with mitochondrial release and caspase-3 activation, respectively. Numerous terminal deoxynucleotidyl transferase-mediated DNA nick end-labelingpositive neurons contained p18 or p20 (65 and 80%, respectively), thereby supporting the idea that cells undergoing cell death contain both processed caspases. Our data are consistent with the idea that transient spinal cord ischemia induces the formation of a death-inducing signaling complex, which may participate in caspase-8 activation and sequential caspase-3 cleavage. Death receptors as well as downstream caspases may be useful therapeutic targets for limiting the death of cells in spinal cord. Key words: caspase-8; caspase-3; spinal cord ischemia; Fas; DISC; cell deathSpinal cord injury after trauma or cerebral ischemia is a major cause of morbidity and mortality (Kouchoukos, 1991;Von Oppel et al., 1994;Cheshire et al., 1996). Many injured cells die by a necrotic mechanism characterized by disruption of nuclear and cell membranes and disintegration of cytoplasmic organelles (Garcia et al., 1995). Some cells die by a mechanism resembling apoptosis, as evidenced by caspase activation (Hara et al., 1997;Endres et al., 1998;Namura et al., 1998;Springer et al., 1999).Caspases are important mediators of ischemic cell death. Procaspase-8 [Fas-associated death domain protein (FADD)-like interleukin-1␤ converting enzyme or MORT1-associated CED-3 homolog] is a 55 kDa initiator caspase (Boldin et al., 1996;Fernandes-Alnemri et al., 1996;Muzio et al., 1996). Procaspase-8 can process itself after ligation of the Fas-tumor necrosis factor family of death receptors (Kischkel et al., 1995;Los et al., 1995;Medema et al., 1997). Fas, a 45 kDa membrane receptor, forms a death-inducing signaling complex (DISC) with an adaptor protein, FADD, and procaspase-8 (Nagata and Goldstein, 1995; Nagata, 1997). Active caspase-8 initiates downstream cleavage of caspase-3 by direct or mitochondrial-dependent mechanisms via BH3 interacting death domain agonist cleavage, leading to apoptosis (Kuwana et al., 1998;Stennicke et al., 1998). In addition, activated caspase-3 may...

Adrenomedullin (AM) is a multifunctional peptide vasodilator that signals through a G-protein-coupled receptor when the receptor, called calcitonin receptor-like receptor (CL), is associated with a receptor activity-modifying protein 2 (RAMP2). We demonstrated previously that haploinsufficieny for each of these genes led to reduced maternal fertility, and that even a modest genetic reduction of AM peptide caused maternal defects in implantation, placentation, and fetal growth. Here, we further demonstrate that Adm(+/-) female mice displayed reduced pregnancy success rates that were not caused by defects in folliculogenesis, ovulation, or fertilization. The poor fertility of Adm(+/-) female mice could not be rescued by transfer of wild-type blastocysts, which suggested an underlying defect in uterine receptivity. In fact, we found that Adm, Calcrl, and Ramp2 gene expressions are tightly and spatiotemporally regulated in the luminal epithelial cells of the uterus during the estrus cycle and the peri-implantation period. RAMP3, which also generates an AM receptor when associated with CL, had a diametrically opposite expression pattern than that of Adm, Calcrl, and Ramp2 and was most robustly induced in the stroma of the uterus. Finally, we discovered that Adm(+/-) female mice have a substantially reduced number of pinopodes on the uterine luminal epithelial surface, which is indicative and possibly causative of the poor uterine receptivity. Taken together, our studies identify a new class of pharmacologically tractable proteins that are involved in establishing uterine receptivity through the regulation of pinopode formation.

We characterized intrinsic and extrinsic specification of progenitors in the lateral and medial ganglionic eminences (LGE and MGE). We identified seven genes whose expression is enriched or restricted in either the LGE: Boc, Fzd8, Ankrd43 and Ikzf1, or MGE: Mbip, Zswim5, and Adamts5. Boc, Fzd8, Mbip and Zswim5 are apparently expressed in LGE or MGE progenitors, while the remaining three are seen in the post-mitotic mantle zone. Relative expression levels are altered and regional distinctions are lost for each gene in LGE or MGE cells propagated as neurospheres; indicating that these newly identified molecular characteristics of LGE or MGE progenitors depend upon forebrain signals not available in the neurosphere assay. Analyses of Pax6Sey/Sey, Shh−/−, and Gli3XtJ/XtJ mutants suggests that LGE and MGE progenitor identity does not rely exclusively upon previously established forebrain-intrinsic patterning mechanisms. Among a limited number of additional potential patterning mechanisms, we found that extrinsic signals from the frontonasal mesenchyme are essential for Shh and Fgf8-dependent regulation of LGE and MGE genes. Thus, extrinsic and intrinsic forebrain patterning mechanisms cooperate to establish LGE and MGE progenitor identity, and presumably their capacities to generate distinct classes of neuronal progeny.

The homozygous mouse mutant weaver exhibits a massive loss of cerebellar granule neurons postnatally. The death of these cells is associated with a single amino acid mutation in the G protein-activated inwardly rectifying potassium channel, Girk2. Evidence suggests that both the mutated Girk2 channel and the calcium channel-associated N-methyl-D-aspartate receptor play important roles in the apoptotic death of weaver cerebellar granule cells, but the downstream events associated with this process are unknown. In this study, we demonstrate that the consequences of the mutation result in caspase activation. In addition, our results show that caspase inhibition in vivo decreases caspase activation and granule cell apoptosis and significantly improves behavioral deficits associated with the weaver's phenotype.